Chemotherapeutic Activities of New η6-p-Cymene Ruthenium(II) and Osmium(II) Complexes with Chelating SS and Tridentate SNS Ligands

A series of new chelating bidentate (SS) alkylimidazole-2-thione-Ru(II)/Os(II) complexes (3ai, 3aii, 3aiii, 3bii/4aiii, 4bi, 4bii), and the tridentate (SNS) pyridine-2,6-diylimidazole-2-thione-Ru(II)/Os(II) complexes (5bi, 5civ/6bi, 6ci, 6civ) in the forms [MII(cym)(L)Cl]PF6 and [MII(cym)(L)]PF6 (M = Ru or Os, cym = η6-p-cymene, and L = heterocyclic derivatives of thiourea) respectively, were successfully synthesized. Spectroscopic and analytical methods were used to characterize the complexes and their ligands. Solid-state single-crystal X-ray diffraction analyses revealed a “piano-stool” geometry around the Ru(II) or Os(II) centers in the respective complexes. The complexes were investigated for in vitro chemotherapeutic activities against human cervical carcinoma (HeLa) and the non-cancerous cell line (Hek293) using the MTT assay. The compounds 3aii, 5civ, 5bi, 4aiii, 6ci, 6civ, and the reference drug, 5-fluorouracil were found to be selective toward the tumor cells; the compounds 3ai, 3aiii, 3bii, 4bi, 4bii, and 6bi, which were found not to be selective between normal and tumor cell lines. The IC50 value of the tridentate half-sandwich complex 5bi (86 ± 9 μM) showed comparable anti-proliferative activity with the referenced commercial anti-cancer drug, 5-fluorouracil (87 ± 15 μM). The pincer (SNS) osmium complexes 6ci (36 ± 10 μM) and 6civ (40 ± 4 μM) were twice as effective as the reference drug 5-fluorouracil at the respective dose concentrations. However, the analogous pincer (SNS) ruthenium complex 5civ was ineffective and did not show anti-proliferative activity, even at a higher concentration of 147 ± 1 μM. These findings imply that the higher stability of the chelating (SS) and the pincer (SNS) ligand architectures in the complexes improves the biological (anti-proliferative) activity of the complexes by reducing the chance of ligand dissociation under physiological conditions. In general, the pincer (SNS) osmium complexes were found to be more cytotoxic than their ruthenium analogues, suggesting that the anti-proliferative activity of the imidazole-2-thione-Ru/Os complexes depends on the ligand’s spatial coordination, the nature of the metal center, and the charge of the metal complex ions.

Half-sandwich arene-Ru(II) complexes with various co-ligands have lately attracted intense scientific interest due to their potential as chemotherapeutics agents [18] and promising alternatives to the potent Pt(II) complex, cisplatin, and its oxaliplatin analogues [19,20].An arene substituent (herein represented by p-cymene) on a metal complex has an advantage as a non-labile ligand and is known to stabilize the Ru metal center in its reactive oxidation state of +2 under physiological conditions [21].Furthermore, experimental evidence shows that the corresponding Ru(II) complexes have kinetic properties similar to Pt(II) complexes [22].In this vein, efforts have been made by researchers to improve the biological activities of metal complexes by using chelating sulfur-containing ligands to achieve rigidity and limit lability of the complexes [23].Related studies have demonstrated that complexes containing benzimidazole-thione ligands of the form: [M II/III (cym/Cp*)Cl]PF 6 (M = Ru, Os, Rh, or Ir; cym = η 6 -p-cymene and Cp* = η 5 -pentamethylcyclopentadienyl) were active anti-proliferative agents in four cell lines [3].However, the less kinetically labile Os and Ir derivatives were more potent than their Ru and Rh analogues [3].Similarly, the design of the imidazole-2-thione ligand framework in this series of new complexes is aimed at taking advantage of its chelating effect and thereby maintaining the +2 oxidation state (with profound physiological effects) on metal (Ru and Os) centers.Hence, the overall object of the current work is to observe their effects on the chemotherapeutic activities of the complexes and the possible activity-structure relationship due to the varying lengths of the spacer and the donor groups in the ligand framework.
Ruthenium and osmium complexes have similar structures and action mechanisms in anti-cancer studies [24,25].However, osmium complexes have a slower ligand exchange rate and are less reactive than the ruthenium analogues [26,27].This suggests that osmium complexes may have greater anti-cancer activities as they can reach the target cell unperturbed, with the ligands still coordinated to the osmium center.Related reviews have shown various works on the popularity of NHC-metal complexes based on the coinage metals and those with the PGM group metals, including Ru [28].However, to the best of our knowledge, such activities based on new imidazole-2-thione Ru or Os complexes are still unreported.
Recently, our group reported on some half-sandwich arene-Ru(II) and Os(II) complexes with N,N -chelating ligands, which were more active against cancer cells than the positive control (anti-cancer drug, 5-fluorouracil) [29][30][31].In further exploring the chemotherapeutic potential of Ru and Os complexes, we herein report for the first time the synthesis, characterization, and in vitro anti-proliferative activities against cervical cancer (HeLa cell line) of a series of new half-sandwich Ru and Os complexes, which are distinguishable by their methylene-, ethylene-, and pyridine-bridged bis(thione) ligands.The chemotherapeutic selectivities of these metal complexes were evaluated using the non-tumor HEK293 (human embryonic kidney) cell line, and a comparison of activities was made with reference to the positive control drug, 5-fluorouracil.
Molecules 2024, 29, x FOR PEER REVIEW 3 of 18 bidentate (SS) alkylimidazole-2-thione ligands (1ai, 1aii, 1aiii, 1bi, 1bii) and the tridentate (SNS) pyridine-2,6-diylimidazole-2-thione ligands (2bi, 2ci, 2civ) were obtained as pale yellow, orange-yellow or colorless solids in moderately high yields, and their identities were confirmed using NMR, FTIR, and HR-MS spectroscopies.The conversion of the imidazolium group into a thione resulted in the loss of the characteristic azolium proton singlet resonance signal at about 10 ppm in the 1 H-NMR spectra of all the imidazole-2-thione ligands [4,5,32,36].Further, the observed far downfield resonance signal at around δ 161.6-163.9ppm in the 13 C-NMR spectra of all the synthesized imidazole-2-thione ligands and the loss of the imidazolium carbon peak (initially at around 143 ppm) from the derivatized salt, confirmed the formation of the C=S group.In the infrared spectra, three bands consistently appeared in the regions: 1526-1688 cm −1 , 1070-1190 cm −1 , and 510-723 cm The new brown half-sandwich imidazole-2-thione-ruthenium complexes 3ai, 3aii, 3aiii, 3bii, 5bi and 5civ and their analogous yellow half-sandwich imidazole-2-thione-osmium complexes 4aiii, 4bi, 4bii, 6bi, 6ci, and 6civ were obtained by stirring 2 molar equivalents of the respective alkyl/pyridine-2,6-diyl-bridged imidazole-2-thione ligands with [Ru(pcymene)(µ-Cl)Cl]2 and [Os(p-cymene)(µ-Cl)Cl]2 in DCM for 16 h at room temperature [37].The solution from each system was then poured into an excess aqueous methanolic solution of KPF6.This resulted in the formation of either the brown or yellow precipitates of the respective complexes.These were then vacuum-filtered and thoroughly washed with water and ethyl ether.Subsequent drying in vacuo afforded the complexes as air and moisture-stable solids in moderate yields (Scheme 2).All the afforded complexes are soluble in aprotic polar solvents like DMSO, MeCN, DMF, and CH3COCH3.They were all characterized by NMR and FTIR spectroscopy as well as HR-MS.All complexes had sharp melting points.Suitable The new brown half-sandwich imidazole-2-thione-ruthenium complexes 3a i , 3a ii , 3a iii , 3b ii , 5b i and 5c iv and their analogous yellow half-sandwich imidazole-2-thione-osmium complexes 4a iii , 4b i , 4b ii , 6b i , 6c i , and 6c iv were obtained by stirring 2 molar equivalents of the respective alkyl/pyridine-2,6-diyl-bridged imidazole-2-thione ligands with [Ru(pcymene)(µ-Cl)Cl] 2 and [Os(p-cymene)(µ-Cl)Cl] 2 in DCM for 16 h at room temperature [37].The solution from each system was then poured into an excess aqueous methanolic solution of KPF 6 .This resulted in the formation of either the brown or yellow precipitates of the respective complexes.These were then vacuum-filtered and thoroughly washed with water and ethyl ether.Subsequent drying in vacuo afforded the complexes as air and moisture-stable solids in moderate yields (Scheme 2).All the afforded complexes are soluble in aprotic polar solvents like DMSO, MeCN, DMF, and CH 3 COCH 3 .They were all characterized by NMR and FTIR spectroscopy as well as HR-MS.All complexes had sharp melting points.Suitable single crystals of 3a i-iii , 3b ii , 4a iii , 5c iv , and 6c i were further used to obtain solid-state X-ray crystallographic data for the complexes.
The 1 H NMR spectra of these complexes showed resonance signals around δ 2.69-2.90 and 5.32-6.37ppm, due to the isopropyl protons CH(CH3)3 and aromatic protons η 6 -C6H4 of the p-cymene moiety in the CD3CN solution, respectively.The resonance signals corresponding to the backbone imidazole (olefinic) protons in the imidazole-2-thione ligand moieties appeared at around δ 6.96-7.68ppm in the spectra of the complexes.The coordination of the ligands to the metal centers caused the signals of the methylene and ethylene protons bridging the thione groups and methylene protons in the lutidyl backbone to become diastereotopic [3,38].This suggested a twisted conformation in the structure of the complexes, as reported for similar complexes bearing a pincer-like architecture [38].The 13 C NMR spectra of all the complexes gave a characteristic slight upfield shift of the resonance signal corresponding to the C=S group to the range δ 152.2-161.6 ppm.This is typical and is attributed to the formation of the S-M bond in the complex molecules [4,34,37,39].
The infrared spectra of the free ligands consisted of υ(C=C)+υ(C=N) [40][41][42] bands in the regions 1639-1688 cm −1 and 1526-1599 cm −1 .FTIR spectra of the corresponding complexes showed a collapse of the peaks in the former region, with a corresponding slight blue shift in the peaks of the latter region.This suggested a decrease in the delocalization of π-electrons within the ligand's heterocyclic moiety.This, consequently, led to the weakening of the υ(C=N) contribution upon coordination.The appearance of a sharp peak between 820-830 cm −1 was ascribed to υ(P-F) [29,43,44], and this confirmed the presence of the PF6 − counter ion in the structure of the complexes.ESI-MS analyses were further used to confirm the identity of the complexes.The ESI-mass spectra of all the complexes Scheme 2. Synthesis of chelating bidentate (SS) alkylimidazole-2-thione-Ru(II)/Os(II) complexes, 3a i , 3a ii , 3a iii , 3b ii /4a iii , 4b i, 4b ii ; and the tridentate (SNS) pyridine-2,6-diylimidazole-2-thione-Ru(II)/Os(II) complexes, 5b i , 5c iv /6b i , 6c i , 6c iv .
The 1 H NMR spectra of these complexes showed resonance signals around δ 2.69-2.90 and 5.32-6.37ppm, due to the isopropyl protons CH(CH 3 ) 3 and aromatic protons η 6 -C 6 H 4 of the p-cymene moiety in the CD 3 CN solution, respectively.The resonance signals corresponding to the backbone imidazole (olefinic) protons in the imidazole-2-thione ligand moieties appeared at around δ 6.96-7.68ppm in the spectra of the complexes.The coordination of the ligands to the metal centers caused the signals of the methylene and ethylene protons bridging the thione groups and methylene protons in the lutidyl backbone to become diastereotopic [3,38].This suggested a twisted conformation in the structure of the complexes, as reported for similar complexes bearing a pincer-like architecture [38].The 13 C NMR spectra of all the complexes gave a characteristic slight upfield shift of the resonance signal corresponding to the C=S group to the range δ 152.2-161.6 ppm.This is typical and is attributed to the formation of the S-M bond in the complex molecules [4,34,37,39].
The infrared spectra of the free ligands consisted of υ(C=C)+υ(C=N) [40][41][42] bands in the regions 1639-1688 cm −1 and 1526-1599 cm −1 .FTIR spectra of the corresponding complexes showed a collapse of the peaks in the former region, with a corresponding slight blue shift in the peaks of the latter region.This suggested a decrease in the delocalization of π-electrons within the ligand's heterocyclic moiety.This, consequently, led to the weakening of the υ(C=N) contribution upon coordination.The appearance of a sharp peak between 820-830 cm −1 was ascribed to υ(P-F) [29,43,44] Single crystals of the complexes 3a i , 3a ii , 3a iii , 3b ii , 4a iii , 5c iv , and 6c i that were suitable for X-ray diffraction analysis were grown via a slow diffusion of diethyl ether into a saturated acetonitrile solution of each of the complexes.Table 1 summarizes the crystallographic data for the compounds and Table 2 gives the selected bond lengths and angles.The traditional piano-stool (half-sandwich) configurations were observed for all complexes (Figures 1 and 2); where the p-cymene acted as the seat along with the SS-chelating thione and chlorido or acetonitrile ligands serving as the legs of the stool (Figure 1: 3a i-iii , 3b ii ; and Figure 2: 4a iii ).The data revealed that during crystallization, the chlorido ligand in 3a i was substituted with an acetonitrile solvent molecule, resulting in a doubly charged complex cation and two PF 6 − counter ions (Figure 1).As expected, the average Ru-S distance (2.3964 Å) in 5c iv is comparatively shorter than those recorded for the other Ru complexes: 3a i (2.4419 Å), 3a ii (2.4566 Å), 3a iii (2.4562 Å), and 3b ii (2.440 Å) (Table 2).This is attributed to the strong pincer-type bonding mode in the 2c i ligand framework and is within the range of the bond length reported for similar compounds [37].The Ru-N bond length, 2.0710( 16) Å in 3a i (Table 2) formed between the ruthenium and the acetonitrile nitrogen (Figure 1) is shorter than the reported value of 2.044(3) Å in an analogous compound [45].On the other hand, the Ru-Cl bond lengths in 3a ii , 3a iii , and 3b ii are observed within the range 2.4018(7)-2.4072(4)Å (Table 2), but are shorter than the reported range of 2.423(12)-2.420(19)Å observed in similar compounds [39].The average C-S bond length in 6c i (1.7030 Å) is also shorter than those in 3a i-iii , 3b ii , 4a ii , 5c iv (Table 2), or as observed in a reported analogue (i.e., 1.7395 Å) [39].However, the recorded C-S bond length in the complex 6c i is longer than the value of 1.6935 Å observed in other similar compounds [37].The S-Ru-S bond angle of 84.700(4) • in the pincer (SNS) Ru complex 5c iv is smaller than those observed in the chelating (SS) Ru complexes 3a i-iii and 3b ii (Table 2).In this same vein, the value is also smaller than the previously reported 85.56(3) • [37] and 93.63 (10) • [39] for similar compounds.As expected, the average Ru-S distance (2.3964 Å) in 5civ is comparatively shorter than those recorded for the other Ru complexes: 3ai (2.4419 Å), 3aii (2.4566 Å), 3aiii (2.4562 Å), and 3bii (2.440 Å) (Table 2).This is attributed to the strong pincer-type bonding mode in the 2ci ligand framework and is within the range of the bond length reported for similar compounds [37].The Ru-N bond length, 2.0710 (16) Å in 3ai (Table 2) formed between the ruthenium and the acetonitrile nitrogen (Figure 1) is shorter than the reported value of 2.044(3) Å in an analogous compound [45].On the other hand, the Ru-Cl bond lengths in 3aii, 3aiii, and 3bii are observed within the range 2.4018(7)-2.4072(4)Å (Table 2), but are shorter than the reported range of 2.423(12)-2.420(19) Å observed in similar compounds [39].The average C-S bond length in 6ci (1.7030 Å) is also shorter than those in 3ai-iii, 3bii, 4aii, 5civ (Table 2), or as observed in a reported analogue (i.e., 1.7395 Å) [39].However, the recorded C-S bond length in the complex 6ci is longer than the value of 1.6935 Å observed in other similar compounds [37].The S-Ru-S bond angle of 84.700(4)° in the pincer (SNS) Ru complex 5civ is smaller than those observed in the chelating (SS) Ru complexes 3ai-iii and 3bii (Table 2).In this same vein, the value is also smaller than the previously reported 85.56(3)° [37] and 93.63(10)° [39] for similar compounds.
The average Os-S bond length (2.4483 Å) in the chelating (SS) osmium complex, 4aiii is similar to the Ru-S bond distance (2.4562 Å) in the isostructural ruthenium complex 3aiii.This demonstrates that these complexes are nearly identical in their three-dimensional structure (Figures 1 and 2; and Table 2).The average Os-S bond length (2.4009 Å) in the pincer (SNS) Os complex 6ci is shorter than the recorded 2.4483 Å in the related chelating (SS) Ru complex, 4aiii (Table 2).This is attributed to the strong pincer (SNS) type bonding mode of the ligand, 2civ.The said value is also shorter than the 2.4401 Å in the previously reported [Os(H)(CO)(PPh3){H2B(mt)2}] [46].Similarly, the 2.4027(12) Å Os-Cl bond length in 4aiii is shorter than the 2.4110(5) Å reported for the related half-sandwich osmium complex [Os(cym)(L)Cl]PF6, where L represents heterocyclic derivatives of thiourea [3].However, this value is comparable to 2.4030(12) Å for the Ru-Cl bond length in the isostructural ruthenium complex 3aiii (Table 2; and Figures 1 and 2).Similarly, there is minimum variation in the bond length between the metal center and pyridine nitrogen, The average Os-S bond length (2.4483 Å) in the chelating (SS) osmium complex, 4a iii is similar to the Ru-S bond distance (2.4562 Å) in the isostructural ruthenium complex 3a iii .This demonstrates that these complexes are nearly identical in their three-dimensional structure (Figures 1 and 2; and Table 2).The average Os-S bond length (2.4009 Å) in the pincer (SNS) Os complex 6c i is shorter than the recorded 2.4483 Å in the related chelating (SS) Ru complex, 4a iii (Table 2).This is attributed to the strong pincer (SNS) type bonding mode of the ligand, 2c iv .The said value is also shorter than the 2.4401 Å in the previously reported [Os(H)(CO)(PPh 3 ){H 2 B(mt) 2 }] [46].Similarly, the 2.4027(12) Å Os-Cl bond length in 4a iii is shorter than the 2.4110(5) Å reported for the related halfsandwich osmium complex [Os(cym)(L)Cl]PF 6 , where L represents heterocyclic derivatives of thiourea [3].However, this value is comparable to 2.4030(12) Å for the Ru-Cl bond length in the isostructural ruthenium complex 3a iii (Table 2; and Figures 1 and 2).Similarly, there is minimum variation in the bond length between the metal center and pyridine nitrogen, ranging between the 2.1733(18) Å Os-N bond length in 6c i and the 2.172(2) Å for the corresponding Ru-N in the previously reported isostructural ruthenium complex [Ru(p-cymene)(Bmtp)](PF 6 ) 2 [37].The S-Os-S bond angle of 90.94 (6) • in 4a iii is larger than the 86.07(2) • in 6c i .However, this value is comparable to the 91.02(2) • recorded for the S-Ru-S angle in the isostructural ruthenium complex 3a iii (Table 2).

Chemotherapeutic Activities of the Arene-Ru(II)/Os(II) Complexes
Statistical data from the archives of the American Cancer Society indicate that cancer is the second major death-causing disease in the world in recent times.It has accounted for about 17% of reported deaths when compared to other diseases [47].Although researchers have made many efforts and have provided some efficient anti-cancer drugs to the markets, certain persistent drawbacks like poor solubility, drug resistance, and negative side effects due to the toxic nature of the chemotherapeutic drugs have also continuously fostered the development of novel, promising, and highly effective anti-cancer drugs aimed at bringing an end to the disease [48].Despite various approaches being practiced in reducing the risk of cancer, there is still a dire need to develop novel treatment strategies that ensure safety as well as efficacy against this life-threatening disease [49].
Certain heterocycles, especially those derived from azoles like imidazole and triazole, have profound potential as effective pharmacological agents [50].The presence of such heterocycles in the structural motif of drug compounds significantly enhances the bioactive nature of the parent structure.The imidazole moiety is generally found in drugs for fungal infections, but many researchers reported imidazole derivatives as potential anti-cancer agents [51].This has motivated us and other researchers to explore novel imidazole derivatives, like the imidazole-2-thiones that we present herein as viable nontoxic substitutes to the known chemotherapeutic agents [52].Thus, we synthesized and assessed the chemotherapeutic potentials of new imidazole-2-thione-Ru/Os complexes on HeLa cancer cell lines and Hek293 cells.The half-sandwich complexes were evaluated alongside 5-fluorouracil (as a positive control) for in vitro cytotoxicity against the cancerous cell lines by the MTT assay method.
The cytotoxicity of the arene-Ru and arene-Os complexes was investigated against HeLa and Hek293 cells.The former cell is a cervical cancer line, while the latter is a nontumor kidney cell line used to examine tumor selectivity.Compounds 3a ii , 5c iv , 5b i , 4a iii , 6c i , 6c iv , and the reference drug 5-fluorouracil were found to be selective toward the tumor cells, as opposed to compounds 3a i , 3a iii , 3b ii , 4b i , 4b ii , and 6b i , which were found not to be selective between normal cell lines and tumor cell lines (Table 3; entries 1-13).The tridentate half-sandwich complex 5b i (86 ± 9 µM) showed comparable anti-proliferative activity with the referenced commercial anti-cancer drug, 5-fluorouracil (87 ± 15 µM).The pincer (SNS) osmium complexes 6c i (36 ± 10 µM) and 6c iv (40 ± 4 µM) were twice as effective as the reference drug (Table 3; entries 11-13).However, the analogous pincer (SNS) ruthenium complex, 5c iv, was less effective and exhibited a higher IC 50 value of 147 ± 1 µM (Table 3; entry 9).These findings imply that the higher stability of the chelating (SS) and the pincer (SNS) ligand architectures in the complexes improve the biological (anti-proliferative) activity by rendering ligand dissociation less likely to occur under physiological conditions [21,24].The pincer (SNS) osmium complexes were more cytotoxic than their ruthenium analogues.These findings suggest that the slower ligand exchange kinetics in these osmium complexes are essential for high cytotoxic activity [3].These allow for more efficient interactions with biological target molecules.Osmium typically forms coordinative bonds with more inert bonds than in the analogous lighter homolog, ruthenium [27].Due to their relative inertness and sufficient stability under physiological conditions, osmium complexes are considered exciting alternatives to ruthenium-based anti-cancer agents [26].However, there was no apparent general pattern in the reactivity of the two metal centers and the isostructural ligand.For instance, the isostructural ruthenium complex 5c iv (147 ± 21 µM) and osmium complex 6c iv (40 ± 4 µM) complexes showed no similar biological activities (Table 3; entries 9 and 12, respectively).The same was also observed with the ruthenium complex 5b i (86 ± 9 µM) and its osmium analogue 6b i (151 ± 12 µM) (Table 3; entries 8 and 10, respectively).This suggested that the effect of the metal center on the anti-proliferative activity depends on the framework in the ligands and their spatial arrangement in coordination to the metal center.Similar findings from a related study have shown that cytotoxicity is affected by the nature of the metal center, with the less kinetically labile Os derivatives being more potent than their Ru counterparts [3].It was also observed that Os metallodrugs are usually equal to or more potent than their Ru analogues when they contain NO, NN, CN, or SN-bidentate ligands [27].Our findings, reported herein, further suggest that due to the observed structure-activity relationship in the biological anti-proliferative activities of these compounds, Os metallodrugs are also more potent than their Ru analogues when they contain pincer (SNS) tridentate ligands.

X-ray Crystallography
X-ray diffraction data on single crystals of 3a i-iii , 3b ii , 5c iv, and 6c i were collected with a Bruker APEX-II CCD diffractometer equipped with graphite-monochromated MoKα radiation (λ = 0.71073 Å).The crystals were kept at 149.99 K during data collection using Olex2 [62].The structures were solved using Intrinsic Phasing with the SHELXT [63] structure solution program and refined with the SHELXL [64] refinement package using Least Squares minimization.The crystal data and structure refinement information obtained are summarized in Table 1.Crystallographic data for the structures in this article have been deposited with the Cambridge Crystallographic Data Centre with CCDC numbers for compounds 3ai-6ci of 2302988-2302990 and 2321930-2321933.The data can be obtained free of charge at http://www.ccdc.cam.ac.uk/conts/retrieving.html, accessed on 16 February 2024 (or from the Cambridge Crystallographic Data Centre, 12 Union Road Cambridge CB2 1EZ, UK; Fax: +44-1223/336-033; E-mail: deposit@ccdc.cam.ac.uk).

MTT Cell Viability Assay
The cytotoxicity of the compounds was examined in the HeLa and HEK293 cells using the MTT assay [65].Cells were trypsinized, seeded into clear 96 well plates, and incubated at 37 • C overnight to allow cells to attach.The cells were then prepared by replacing the growth medium with a fresh complete medium (EMEM+10% FBS+ 1% Antibiotics).The samples were then added as a DMSO/H 2 O solution to the cells at various concentrations and incubated at 37 • C for 48 h.After that, the growth medium was aspirated and replaced with the medium (0.1 mL) and MTT solution (0.01 mL: 5 mg/mL in PBS), and the cells were incubated for a further 4 h.The medium was then removed and replaced with DMSO (0.1 mL).Absorbance measurements were then taken at 570 nm using a Mindray MR-96A microplate reader (Vacutec, Hamburg, Germany).
, and this confirmed the presence of the PF 6 − counter ion in the structure of the complexes.ESI-MS analyses were further used to confirm the identity of the complexes.The ESI-mass spectra of all the complexes (except for 4 aiii , 4b ii , 5b i , and 6b i ) contained molecular [M−PF 6 ] + ions at m/z values that match calculated values.The spectra of the complexes 4 aiii , 4b ii contained [(M−PF 6 −Cl)] 2+ ions, while those of 5b i and 6b i showed fragmented ions [(M−PF 6 −2CH 3 )] 2+ .The ob-served m/z values were comparable to the expected calculated data.(See the electronic Supplementary Materials).

Table 3 .
Ruthenium and osmium complexes MTT cytotoxicity test results.
a IC 50 values correspond to the concentration of the respective compound needed to cause 50% net cell mortality.